SPECT analysis of <i>in vivo</i> accumulation of Tc-99m-HYNIC-VEGF-c.

<p>VEGF-c (which targets both VEGFR2 and VEGFR3) was tagged with HYNIC chelators and then labeled with Tc-99m-pertechnetate (Tc-99m) and injected intravenously in PTK787 and vehicle treated rats. One hour after injection, SPECT images were obtained using dedicated animal scanner. All PTK787 treated rats showed increased accumulation of Tc-99m-HYNIC-VEGF-c in the tumors (lower panel, arrows) compared to that of vehicle treated tumors (upper panel, arrows).</p

Western blot images and densitometry analysis of western blots.

<p>(<b>A</b>) Expression of different angiogenic factors (left panel) in the vehicle- and PTK787 treated tumors from representative cases at the peripheral (P), central part of the tumors (C) and contralateral brains (B). Note the increased expression of VEGF, SDF-1 and HIF-1α at the peripheral part of PTK787 treated tumors. Right panel shows the densitometry analysis of the blot (normalized to β-actin and contralateral brain). The analysis also confirmed the finding of the blot. Note the patterns of VEGF, SDF-1 and HIF-1α in PTK787 treated tumors which are different from that of vehicle treated tumors. (<b>B</b>) Densitometry analysis of VEGFR2, VEGFR3 and EGFR blot (normalized to β-actin and contralateral brain). Expression of VEGFR2, VEGFR3 and EGFR showed higher normalized values at the peripheral part of the PTK787 treated tumors compared to that of central part and the expression patterns are different in vehicle treated tumors. Please note that PTK787 treated tumors showed lower normalized values of VEGFR2 and EGFR both at the periphery and central parts of the tumors compared to that of corresponding contralateral brain; whereas vehicle treated tumors did not show any changes in the normalized values of VEGFR2 and EGFR compared to that of corresponding contralateral brain. Data are expressed as mean ± SEM, n = 3.</p

Immunohistochemistry of PTK787 and vehicle treated tumor showing expression of VEGF, HIF-1α, SDF-1 and vessel morphology.

<p>Expression of vascular endothelial growth factor (VEGF) (dark brown colored) at different parts of the PTK787 treated and vehicle treated tumors. There were no differences observed in the expression of VEGF on immunohistochemistry at different parts of the tumors treated with either PTK787 or vehicle. However, delineation of vessels using FITC tagged tomato lectin indicated higher number of dilated vessels at the tumor periphery in rats that received PTK787 treatment. These dilated vessels may be indicative of increased permeability, fPV and signal intensity changes in PTK787 treated tumors (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008727#pone-0008727-g001" target="_blank"><b>Figures 1</b></a><b> and </b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008727#pone-0008727-g002" target="_blank"><b>2</b></a>). Increased permeability may also be due to increased VEGF expression. HIF-1α expression was mostly seen in the central part of the vehicle treated tumors (arrows), however, SDF-1 expressions were observed both in PTK787 and vehicle treated tumors (arrows).</p

Immunohistochemistry of PTK787 and vehicle treated tumor showing expression of VEGFR2, VEGFR3 and EGFR.

<p>Immunohistochemistry confirmed the findings of SPECT studies, where PTK787 treated tumors showed increased expression of VEGFR2 and VEFGR3 at the peripheral parts of the tumors, especially around the vessels (arrows) compared to that of vehicle treated tumors (right column). Both PTK787 (lower panel, left column) and vehicle treated (lower panel, right column) showed expression of EGFR in the tumors. Lower panel, middle column show no brown cells in negative control slide.</p

Immunohistochemistry staining with Ki-67 in vehicle treated and melatonin treated tumors.

<p>Images were taken with 40× magnification. There was a decreased cell proliferation in tumors treated with melatonin (*p<0.05). Error bars: ± standard error.</p

SPECT analysis of <i>in vivo</i> accumulation of Tc-99m-HYNIC-VEGF-c.

<p>VEGF-c (which targets both VEGFR2 and VEGFR3) was tagged with HYNIC chelators and then labeled with Tc-99m and injected intravenously in melatonin and vehicle treated mice. One hour after injection, SPECT images were obtained using dedicated animal scanner. Vehicle treated mice showed increased accumulation of Tc-99m-HYNIC-VEGF-c in the mammary tumor (A, Intersection of lines indicate the tumor, with a volume of 865.69 mm³ at the 21th day) compared to that of melatonin treated mammary tumors (B, Intersection of lines indicate the tumor, with a volume of 130.69 mm³ at the 21th day) C. Semi-quantitative analysis of total radioactivity normalized to contralateral muscles showing the intensity of radioactivity in the vehicle and melatonin treated animals.</p

Immunohistochemistry staining with VEGFR2 (arrows) in vehicle treated and melatonin treated tumors.

<p>Images were taken with 40× magnification. A significant decrease was observed at the tumor in melatonin treated tumors compared to vehicle treated tumors (*p<0.05). Error bars: ± standard error.</p

Immunohistochemistry staining with VEGFR3 (arrows) in vehicle treated and melatonin treated tumors.

<p>Images were taken with 40× magnification. Melatonin do not decreased significantly the expression of VEGFR3 (p>0.05). Error bars: ± standard error.</p

Comparison between proteins expression in mammary tumor in animals treated with melatonin or vehicle.

<p>White column  =  vehicle treatment; Black column  =  melatonin treatment. Data are shown as mean ± S.D. *p<0.05, vs. Control.</p

Antitumor effects of melatonin on mammary tumor growth.

<p>Melatonin reduced the tumor growth in breast cancer nude mice. Each point in the curves represents the mean ± SD (control n = 8; melatonin n = 5). The melatonin inhibited tumor growth, *p<0.05 vs Control. # Significant increase in tumor volume on control group at 14 and 21 after tumor implantation and initiation of treatment with vehicle (p<0.05). Detail: Representative samples of mammary tumors developed by MDA-MB-231 cells implantation on the right flank of mice. A, B. Melatonin treated mammary tumors, B. Mammary tumor which regressed with melatonin treatment. C, D. Vehicle treated mammary tumors.</p